Histological and cytological techniques have been used to analyse biopsies and other tissue samples, as an aid to medical diagnosis and research. Cytology is the study of the structure of all normal and abnormal components of cells and the changes, movements, and transformations of such components. Cells are studied directly in the living state or are killed (fixed) and prepared by for example thin layer preparation systems, embedding, sectioning, and/or staining for investigation in bright field, fluorescent or electron microscopes. Histology is the study of groups of specialised cells called tissues that are found in most multi-cellular plants and animals. Histological investigation includes study of tissue and cell death and regeneration and the reaction of tissue and cells to injury, a disease state such as cancer or invading organisms such as HPV (Human Papilloma Virus). Because normal tissue has a characteristic appearance, histological examination is often utilised to identify diseased tissue.
In situ hybridisation (ISH), and Immunohistochemistry (INC) analyses are useful tools in histological diagnosis and the study of tissue morphology. In situ hybridisation (ISH), immunocytochemistry and immunohistochemistry (INC) seek to identify a detectable entity in a sample by using specific binding agents capable of binding to the detectable entity.
A biological sample is in this application to be understood as a biological sample such as histological samples, e.g. tissue and cell specimens, including cell lines, proteins and synthetic peptides, tissues, cell preparations, blood, bodily fluids, blood smears, metaphase spreads, bone marrow, cytology specimens, thin-layer preparations, and specifically biological samples on microscope slides. The biological sample may further suitably be selected from histological material, including formalin fixed and paraffin embedded material, cytological material, fine needle aspirates, cell smears, exfoliative cytological specimens, touch preparations, bone marrow specimens, sputum samples, expectorates, oral swabs, laryngeal swabs, vaginal swabs, bronchial aspirates, bronchial lavage, gastric lavage, blood, urine, and body fluids. Such biological samples may be subjected to various treatments. Further, the biological sample may be suitably selected from non human sources, including virus and fungus swabs, samples taken from medical equipment, veterinary samples and food. Also, samples may be taken from hair, organs, sperm and egg cells as well as cell grown in vitro. The biological samples are preferably from living or post-mortem tissues of Homo sapiens, but not limited to eukaroytic cells. Examples include detection of prokaryotic organisms, such as Escherichia coli 0157 in drinking water.
Slides can be any suitable solid or semi solid support for the biological sample. In particular, the support may be a microscope slide, a micro array, a membrane, a filter, a polymer slide, a chamber slide, a dish, or a Petri dish.
The current invention relates especially—but not exclusively—to in situ hybridisation (ISH). In situ hybridisation is a diagnostic method for characterization and evaluation of genes, chromosomes, cells, cell aggregates, tissues and other biological samples. In situ hybridisation can be used to evaluate and characterize the status, genetic abnormalities and other disease states, such as cancer or disease, caused by infectious organisms. Further, it can be used to characterize cells with respect to infectious agents such as, but not limited to, HPV, HIV (Human Immunodefiency Virus) and HCV (Hepatitis C Virus). Molecular genetic events, such as aneuploidy, gene amplification, gene deletion, RNA expression, RNA transportation, RNA location and chromosome translocations, duplications, insertions, or inversions that are difficult to detect with karyotype analysis, PCR (Polymerase Chain Reaction), or LCR (Ligase Chain Reaction) can be characterized by ISH.
The ISH techniques can have the potential to increase the survival chances of cancer patients by making possible earlier detection of malignancy and more accurate prognostic assessments following tumour surgery. The technique can also be applied to prenatal and postnatal genetic analysis. Furthermore, the technology can be used for simultaneous detection of multiple genetic anomalies in an individual cell, and thereby save assay time and limit specimen requirements.
Non limiting examples of diagnostically important ISH assays include detection of HER-2 (also known as HER-2/neu or c-erbB2), Topo II (breast carcinoma), telomers, EGFr, C-Myc (breast carcinoma), N-Myc (neuroblastoma); translocation probe pairs for BCR/ABL (chronic myelogenous leukemia), EWS (Ewing's sarcoma), C-Myc (Burkitt's lymphoma, T cell ALL), acute myeloid leukemia (AML), myeloproliferative disorders (MPD), Myelodysplastic Syndrome (MDS) and centromeric probes for chromosomes 17, 7, 8, 9, 18, X, and Y. Other examples include the analysis of Epstein-Barr virus, Herpes simplex virus and Human cytomegalo virus, Human papilloma virus, Varizella zoster virus and Kappa and Lambda light chain mRNAs. Yet other examples include the detection and analysis of samples of non-human origin, for example, food borne parasites and disease causing microbes and viruses. More specific examples include:
i) the analysis of HER-2/neu, also known as c-erbB2 or HER-2, which is a gene that has been shown to play a role in the regulation of cell growth. The gene codes for a transmembrane cell surface receptor that is a member of the tyrosine kinase family. HER-2 has been shown to be amplified in human breast, ovarian and other cancers;
ii) the analysis of aneuploidy for chromosomes 3, 7, 17 and loss of the 9p21 locus in urine specimens from patients with transitional cell carcinoma of the bladder;
iii) the detection and quantification of the lipoprotein lipase (LPL) gene located at 8p22 and the C-MYC gene located at the 8q24 region (Two genetic alterations observed in abnormal cells, such as Prostate cancer samples, are gain of 8q24 and 8p21-22 (LPL) loss of heterozygosity.);
iv) the identification and enumeration of chromosome 8 in cells obtained from bone marrow. An association has been made between trisomy 8 and both myeloid blast crisis and basophilia (Trisomy 8 is a prevalent genetic aberration in several specific diseases like Chronic Myelogenous Leukemia (CML), acute myeloid leukemia (AML), and myeloproliferative disorders (MPD).);
v) the analysis of chromosome aneuploidy like translocations of the immunoglobulin heavy chain locus (IGH) located at 14q32 and frequently observed in patients with various hematological disorders (These IGH translocations result in the upregulation of oncogenes due to the juxtaposition of IGH enhancers with these oncogenes.);
vi) the identification of inv(16)(p13q22) where the CBFB gene located in 16q22 is fused to the MYH11 gene located in 16p13, resulting in a chimeric protein product detected in acute myeloid leukemia (AML);
vii) the detection of Human Papilloma Viruses (HPV), which are a group of small DNA viruses (There are more than 90 HPV types. Persistent HPV infection may result in cervical cancer, and has also been associated with other types of cancer, e.g. colon cancer. HPV types are classified according to the risk associated with the development of cervical cancer. Fifteen types are classified as high-risk, and these are detected in more than 99% of all cervical cancers.).
In summary, the in situ Hybridization (ISH) technique is a useful method for the analysis of cells for the occurrence of chromosomes, chromosome fragments, genes and chromosome aberrations like translocations, deletions, amplifications, insertions or inversions associated with a normal condition or a disease. Further, ISH is useful for detection of infectious agents as well as change in levels of expression of RNA.
The ISH techniques should be understood to include, for example, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), Fiber FISH, CGH, chromosome paints and arrays. In the following, the ISH technique and procedures are described in greater detail. ISH uses nucleic acid probes, designed to bind, or “hybridize,” with the target DNA or RNA of a specimen, usually fixed or adhered to a glass slide. DNA, RNA, PNA, LNA or other nucleic acid probes of synthetic or natural origin can be used for the ISH technique. The probes are labelled to make identification of the probe-target hybrid possible by use of a fluorescence or bright field microscope. The probe is typically a double or single stranded nucleic acid, such as a DNA or RNA. It is labelled using radioactive labels such as 31P, 33P or 32S, or non-radioactively, using labels such as digoxigenin, or fluorescent labels, a great many of which are known in the art. The hybrid is often further analysed with computer imaging equipment. Since hybridization occurs between two complementary strands of DNA, or DNA analogues, labelled probes can be used to detect genetic abnormalities, providing valuable information about prenatal disorders, cancer, and other genetic or infectious diseases.
Unlike other molecular DNA-based tests, which require cell lysis to free nucleic acids for analysis, ISH allows analysis of DNA in situ, that is, in its native, chromosomal form within the cell or even the nucleus. This feature permits the analysis of chromosomes, genes and other DNA/RNA molecules of individual cells. For direct-labelled probes, the results are detected by viewing the samples under a fluorescence microscope with appropriate filters. Indirect detection, like CISH, demands additional labelling steps, which typically require streptavidin or antibody-enzyme conjugates or fluorophore-labeled counterparts, and additional washing steps once the probe is bound to the target.
An exemplified general ISH procedure includes one or several of the following sequential procedural steps:
i) Mounting of the biological sample on slides
ii) Baking at elevated temperatures
iii) Dewaxing or deparaffination if necessary
v) Washing
v) Target retrieval at elevated temperature
vi) Denaturing at elevated temperature
vii) Incubation with blocking reagents
viii) Addition of probe mixture to the sample on the slide.
ix) Placing a coverslip over the sample and the probe mix and sealing with rubber cement.
x) Hybridization at elevated temperatures.
xi) Washing at elevated temperatures and removal of coverslip
xii) Air drying and counterstaining
xiii) Visualization according to the instruction for FISH or CISH
xiv) Examination and evaluation in a microscope
In more detail, an exemplified FISH protocol for paraffin embedded tissue sections could include one or several of the following sequential procedural steps:
i) Cutting 2-4 micrometer tumour sections from a block
ii) Mounting on slides
iii) Baking at 60° C. for 30 minutes
iv) Deparaffination using xylene
v) Rehydration by immersing in ethanol/water mixtures
vi) Pre treating by washing with an aqueous buffer for 10 minutes at 95° C.
vii) Pepsin digesting for 10 minutes at ambient temperature
viii) Washing repeatedly
ix) Dehydration in a series of cold ethanol/water mixtures
x) Air drying
xi) Addition of 10 microliter fluorescent labelled DNA or PNA probe mixture per slide
xii) Sealing with a 22 by 22 mm glass coverslip and rubber cement at the edges
xiii) Denaturing at 82° C. for 5 minutes, directly followed by
xiv) Hybridization over night (18 hours) at 45° C.
xv) Removal of the coverslip
xvi) Stringent washing at 65° C. for 10 minutes
xvii) Washing repeatedly with wash buffer
xviii) Dehydration by immersing in a series of cold ethanol/water mixtures
xix) Air drying
xx) Mounting with 10 microliter anti fade solution with DAPI as counter stain
xxi) Sealing with a coverslide
xxii) Examination and evaluation in a fluorescence microscope
The hybridization mixture is typically a complex mixture of many components. Non-limiting examples of components include formamide, water, triton x-100, tween 20, Tris or Phosphate buffer, EDTA, EGTA, polyvinylpyrrolidine, dextran sulfate, Ficoll, or salmon sperm DNA.
Chromogenic in situ hybridization (CISH) uses labelled probes, which can be visualized by the use of immunological staining methods similar to the IHC staining procedures. CISH has some differences compared to FISH techniques: The genetic aberrations may be viewed within the context of tissue morphology—simultaneous examination of histopathology and ISH results. Also, the results may be visualized with a standard bright field microscope, and the chromogenic dye (for example DAB) generated on the slide is permanent with no or little fading of fluorescent signals.
In addition to ISH, the current invention also relates to immunohistochemistry and immunocytochemistry. The general exemplified formalin fixed paraffin embedded (FFPE) immunohisto chemical (IHC) chromogenic staining procedure may involve the steps of: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibody, washing, applying secondary antibody-enzyme conjugate, washing, applying enzyme chromogen substrate, washing, counter staining, cover slipping and microscope examination.
As described above, the sample treatment of the slides is complicated, laborious and uses many different reagents at various temperatures for prolonged periods. It should be understood that under normal conditions only small amount of reagents, 200 μl or even less, are applied to the sample. Thus, the reagent and sample are very easily dried out, especially under high temperatures and at low relative humidity. Many of the procedural steps in ISH, including the denaturing and the hybridization steps are typically done in a humidity chamber. The humidity chamber is a closed or semi closed container in which the slides can be processed and heated. It should be understood that the processing temperature as well as the temperature ramp time—that is, the change of temperature per time unit, is important for both the overall protocol length and the subsequent visualized result. Furthermore, it has been observed that the staining result depends strongly on the humidity during the sample treatment. Also, the morphology can suffer from drying out during the treatment. For example, chromosome spreads are easily ruined due to drying out conditions. During the changes of temperatures the air above the slides will expand or contract. The reduction in pressure during lowering of the temperature will draw in air from the outside, which may be less saturated with water compared to the air above the slide. During heating, air will be pressed out of the space between the slide and the lid. This air will contain moisture, which will escape from the system. Consequently, due to the plurality of fast and repeated changes in temperature, high temperatures for prolonged time and the small space between the slides and the lid, moisture can escape either quickly, or over time, from the system, resulting in a change in the concentration of the reagents applied to the biological sample and thus a change in the protocol, or even drying out of the biological sample.
The absolute humidity is defined as the amount of water in a given volume of gas. The relative humidity is the ratio between the amount of water and the maximum amount of water possible at the given temperature and pressure. The maximum amount of water per volume, and consequently the relative humidity, depends strongly on the temperature, as described by the Clausius-Clapeyron equation. For example, without addition of water in a closed system, 100% relative humidity at 25° C. will correspond to 16.3% at 60° C. and 3.7% at 95° C., indicating the strong dependence of temperature. Even a small change of temperature will change the relative humidity dramatically. For example, a relative humidity of 100% at 80° C. will correspond to only 66.7% at 90° C. in a closed system without addition of moisture.
From the discussion above, it should be clear that precise control of humidity, heating and cooling is essential for obtaining, for example, consistent ISH results. Therefore, without an efficient humidifying system, heating of the slides can result in fast drying out of the reagents or sample.